Putative Role of CFSH in the Eyestalk-AG-Testicular Endocrine Axis of the Swimming Crab Portunus trituberculatus

Simple Summary The crustacean female sex hormone (CFSH) plays a crucial role in the development of secondary sexual characteristics in female Decapoda crustaceans, but its physiological function in males is less reported. In the present study, the CFSH of P. trituberculatus (PtCFSH) was identified, cloned and expressed in the recombinant form. The physiological functions of PtCFSH on the AG and testis were investigated using the RNAi and recombinant protein injection in a series of in vivo and in vitro experiments. It was confirmed that PtCFSH can negatively regulate the spermatogenesis and testicular development via a CFSH-IAG-testis endocrine axis. PtCFSH may also act directly on the testis, dependent or independent of IAG. The signaling system of PtCFSH involves the participation of cAMP, cGMP, and NO, which resembles the MIH signaling pathway. In summary, our results demonstrated the mode of CFSH action in P. trituberculatus. Abstract It has been shown in recent studies that the crustacean female sex hormone (CFSH) plays a crucial role in the development of secondary sexual characteristics in Decapoda crustaceans. However, research on the function of CFSH in the eyestalk-AG-testicular endocrine axis has been inadequate. We cloned and identified a homolog of CFSH, PtCFSH, in this study. RT-PCR showed that PtCFSH was mainly expressed in the eyestalk. A long-term injection of dsPtCFSH and recombinant PtCFSH (rPtCFSH) in vivo showed opposite effects on spermatogenesis-related gene expression and histological features in the testis of P. trituberculatus, and was accompanied by changes in AG morphological characteristics and PtIAG expression. In addition, the phosphorylated-MAPK levels and the expression of several IIS pathway genes in the testis was changed accordingly in two treatments, suggesting that PtCFSH may regulate the testicular development via IAG. The hypothesis was further validated by a mixed injection of both dsPtCFSH and dsPtIAG in vivo. The following in vitro studies confirmed the negatively effects of PtCFSH on AG, and revealed that the PtCFSH can also act directly on the testis. Treatment with rPtCFSH reduced the cAMP and cGMP levels as well as the nitric oxide synthetase activity. These findings provide vital clues to the mechanisms of CFSH action in both the eyestalk-AG-testis endocrinal axis and its direct effects on the testis.


Introduction
The traditional view is that sexual differentiation in crustaceans is a male-dominated process. For example, in crustaceans, a male-specific androgenic gland (AG) determines male sexual differentiation and the maintenance of secondary sexual characteristics; after the removal of the AG, crustaceans undergo feminization [1,2], and vice versa [3]. An essential hormone was later identified as an insulin-like androgenic gland hormone (IAG) in AG. Moreover, the Sagi group have achieved functional sexual reversal by chronically interfering with MrIAG expression in young Macrobrachium rosenbergii [4]. This also induced the cessation of spermatogenesis in the spermathecae and the incomplete development of secondary sexual characteristics. The function of silencing IAG in Cherax quadricarinatu we anesthetized the crabs on ice. All the experiments comply with the requirements of the governing regulation for the use of experimental animals in Zhejiang Province (Zhejiang provincial government order No. 263, released on 17 August 2009, effective from 1 October 2010) and the Animal Care and Use Committee of Ningbo University. Healthy wild adult swimming crabs with a body weight of 100 to 140 g were purchased from the local aquatic market in Xianxiang, Ningbo. Some individuals in the intermolt period were temporarily reared in a capacious 120 L tank bottom containing 30 cm of fine sand and shelter in our laboratory; the crabs were fed and their water was changed periodically until the 20-day (long-term) and 3-day (short-term) treatment of RNAi and the in vivo recombinant proteins injection was completed. Other crabs were collected for gene cloning and in vitro treatment, respectively. All samples were stored in RNA preservation fluid (Cwbiotech, Taizhou, China) at −80 • C until RNA extraction.
In addition, the testis and AG-related tissues were sampled from the crab and cut into 3-5 mm pieces and fixed in 4% Paraformaldehyde Fix Solution for 24 h at 4 • C. Then, the tissues were dehydrated with serial ethanol, cleared with serial xylene, and finally embedded in paraffin. Tissues were cut into 5-7 µm sections for hematoxylin and eosin (H&E) staining. Next, 30 to 50 mg of testis tissue was placed in a 1.5 mL EP tube containing 300 microliters of PBS and (Protease and phosphatase inhibitor cocktail for general use, 50×) (Beyotime, Shanghai, China), after which the homogenate was centrifuged at 8000 g for 10 min at 4 • C. The supernatant was sucked and stored at −80 • C for western blotting.

Extraction of Total RNA and cDNA Synthesis
Total RNA was isolated from different samples using the RNA-Solv ® reagent (Omega Bio-tek, Norcross, GA, USA) according to the manufacturer's instructions. RNA concentrations were determined using a NanoDrop 2000 UV Spectrophotometer (Thermo Fisher Scientific, Cheshire, UK). The genomic DNA removed with (10× gDNA Remover Mix) and the first strand of cDNA was synthesized using a HiFiScript gDNA Removal cDNA Synthesis Kit (Cwbiotech, Taizhou, China) according to the manufacturer's protocol, followed by storage at −80 • C until use.

Gene Cloning and Bioinformatics Analyses
A keyword-based screening of our RNAseq library (unpublished) yielded a short transcript homologue of the CFSH; this was validated by reverse transcription polymerase chain reaction (RT-PCR) and elongated by the 3 and 5 rapid amplification of cDNA ends (RACE) (Clontech, California, USA) using cDNA generated from muscle-extracted RNA. Table 1 shows the primers used in the cDNA clone. The following program was used for each PCR amplification: initial denaturation at 94 • C for 5 min, followed by 35 cycles of 94 • C for 30 s, 55 • C for 30 s and 72 • C for 90 s, with a final elongation at 72 • C for 10 min. All PCR products were analyzed using 1% agarose gel electrophoresis, then ligated with the pMD19-T vector (Takara, Kyoto, Japan) and transformed into competent Escherichia coli cells. After transformation, three positive clones were picked for sequencing (BGI Tech, Shenzhen, China). The amplified sequences from 3 and 5 RACE were assembled with the partial cDNA sequence using Vector NTI 10.0 software, and the open reading frame (ORF) was identified using ORF finder (http://www.ncbi.nlm.nih.gov/gorf/) (accessed on 22 January 2022). Predictions of the conserved domains were performed using the SMART program (http://smart.emblheidelberg.de/) (accessed on 22 January 2022). The 3D structure of the PtCFSH protein was predicted by the Alphafold2 web server (https:// alphafold.ebi.ac.uk/) (accessed on 15 February 2022) and edited with the PyMOL Software (https://pymol.org/2/) (accessed on 27 February 2022). A multiple sequence alignment and phylogenic analysis was performed using ClustalW1.8 and MEGA7.0 (https://www. megasoftware.net/) (accessed on 28 February 2022).

Semiquantitative PCR
To detect the expression levels of PtCFSH in different tissues, primers PtCFSH-RTF/RTR (Table 1) were designed to generate a 212-bp fragment. Semiquantitative PCR was performed using β-actin as an internal reference to adjust the number of cDNA templates. Amplification was performed with the following program: denaturation at 94 • C for 5 min, followed by 35 cycles of 94 • C for 30 s, 55 • C for 30 s and 72 • C for 90 s, with a final elongation at 72 • C for 10 min. The products were assessed by electrophoresis on 1.5% agarose gel.

RNA Interference and Recombinant Protein Preparation and Injection
The double-strand RNA for PtCFSH, PtIAG, and green fluorescent protein (GFP) were synthesized as previously described [24]. The plasmid pGEX-4t-2 was digested with restriction endonucleases SmaI and XhoI in order to generate a linearized vector. Primers PtCFSH-GST-F/R with 21-bp (Table 1) extensions were designed and synthesized, which were homologous to the linearized vector 5 and 3 ends to generate the amplification products. The purified PCR product was inserted into the corresponding linearized vector by setting up an in-fusion cloning reaction using the ClonExpress II One Step Cloning Kit (Vazyme, Nanjing, China) and then transformed into E. coli cells. The confirmed recombinant plasmids were extracted using the E.Z.N.A. Plasmid Mini Kit I (Omega Bio-Tek) and then transformed into E. coli Rosetta-gami cells to express the rGST-CFSH protein. The vector pGEX-4t-2 as a control was also expressed as the GST tag protein (rGST). The recombinant proteins were purified using the GST-tag Purification Resin (Beyotime, Shanghai, China) and verified three times by SDS-PAGE.
For long-term treatments in vivo, the double-strand RNA of CFSH (dsCFSH) and the recombinant CFSH protein (rCFSH) were injected into crabs at 3 ug/g every three days for 20 days, with dsGFP and rGST serving as controls. The testes and AGs were collected to observe tissue changes. In the short-term RNAi treatment in vivo, dsCFSH, dsIAG, and dsGFP were all injected into different individuals at a dose of 3 ug/g dose, and samples were collected after 48 h. For the in vitro experiments, the desired eyestalks, AGs and testis tissues were all precultured in M199 medium for 1 h. Afterwards, dsCFSH and dsGFP were added at a dose of 3 µg/g dose [20] to Petri dishes (Jet Biofil, Guangzhou, China) containing the eyestalks and AGs, and then co-cultured at 26 • C for 8 h. Additionally, rCFSH and rGST were added to the dishes containing AGs or testes at doses of 10-6 M, respectively. Incubation was continued at 26 • C for 8 h. All samples were collected for qPCR assay and western blot analysis.

Quantitative Real-Time PCR
We determined the relative gene expression levels using quantitative real-time PCR (qPCR). The template cDNA was prepared as described above and then amplified with qPCR primers (Table 1). PCR was carried out using the SYBR ® Premix Ex Taq™ II Kit (Takara, Kyoto, Japan) according to the manufacturer's instructions. PCR conditions were as follows: 95 • C for 2 min, followed by 40 cycles of 95 • C for 15 s and 56 • C for 20 s. Relative mRNA expression levels were normalized to β-actin mRNA expression [25] and calculated using the comparative Ct (2 −∆∆Ct ) method [26].

Western Blot Analysis
For the Western blot assay, the protein concentration of the testis was measured using the BCA Protein Assay Kit (Cwbiotech, Taizhou, China). For SDS-PAGE, 30 mg of protein from each sample was used, followed by electrophoretic transfer to a 0.45 βm PVDF membrane using a Mini Trans-Blot (Bio-Rad, California, USA). After blocking with Y-Tec Rapid Blocking Buffer (Yoche, Shanghai, China) at room temperature for 1 h, the membranes were incubated with Phospho-p44/42 mos/mitogen-activated protein kinase (MAPK) (Erk1/2) or p44/42 MAPK (Erk1/2) (Cell Signaling Technology, Danvers, MA, USA) monoclonal antibodies diluted to 1:1000 in (Y-Tec Stabilizing Antibody Diluent) (Yoche, Shanghai, China) overnight at 4 • C. The membranes were washed three times with TBST (20 mM Tris-HCl, 150 mM NaCl, and 0.05% Tween-20) and then incubated with β-Tubulin Rabbit IgG polyclonal antibody (CST) diluted to 1:2000 in Y-Tec Rapid Blocking Buffer (Yoche, Shanghai, China) at room temperature for 1 h. After washing three times with TBST for 10 min each, the membrane was incubated in a Western Bright ECL substrate (Advansta, California, USA) before exposure to ChemiScope 6000 (CLiNX, Shanghai, China). The protein bands were quantified using the ChemiScope figure software package, and the results were derived from the statistical analysis of three independent experiments.

Measurement of the Level of cAMP, cGMP, and NOS in Testis
The testis was collected at 5, 15 min and 6 h after rCFSH (10 −6 M) and forskolin (10 −5 M) treatment (three replicates per treatment). The cAMP levels and the cGMP levels of the sampled testis at 5 and 15 min were measured using the cAMP ELISA kit (Nanjing Jiancheng Bioengineering Institute of China, Nanjing, China) and cGMP ELISA kit (Nanjing Jiancheng Bioengineering Institute of China, Nanjing, China), respectively. Samples collected after 6 h of treatment were used to detect changes in nitric oxide synthase (NOS) levels using a NOS-typed assay kit (Nanjing Jiancheng Bioengineering Institute of China, Nanjing, China).

Histological Observation of the Testis of RNAi and Recombinant Protein Injection
At the end of the long-term RNAi and recombinant protein injection experimental period, crabs from four groups with a body width of 12.2-14.6 cm were collected. Testis were dissected from the individuals, cut into 3 mm 3 pieces, and fixed in PFS for 24 h at 4 • C. After dehydration in gradient ethanol, they were embedded in paraffin. Paraffin-embedded AGs and testis tissues were sectioned into slices of 5-7 µm. The sections were then stained with H&E and observed under a Nikon Eclipse 80i microscope (Nikon, Tokyo, Japan).

Conceptual Diagrams of Hypothetical Pathways
This hypothetical pathway was constructed with reference to the classical insulin pathway and the general neuropeptide signaling pathway [27]. In this paper, we complete the hypothetical pathway by figdraw.

Statistical Analysis
All data are expressed as mean ± SEM and were subjected to a one-way analysis of variance (ANOVA), followed by Student's t-test or Tukey's multiple-group comparison test. The data were analyzed using SPSS 24.0 software. Significant differences were accepted at p < 0.05. Asterisk denotes statistically significant differences: * p < 0.05; ** p < 0.01; *** p < 0.001.

Molecular Characterization and Tissue Distribution of PtCFSH
We obtained a 1241 bp PtCFSH cDNA (Genbank accession number: ON929327) comprising a 19 bp 5 -untranslated region (UTR), 678 bp ORF, and 542 bp 3 -UTR with a poly (A) tail ( Figure 1). A sequence analysis of the 678 bp ORF revealed 225 amino acids with a 24 aa signal peptide, 31 aa CFSH-precursor-related peptides, a 2 aa cleavage site and a 166 aa interleukin-17 domain containing eight cysteines, forming four disulfide bonds ( Figure 1A,B). The three-dimensional structure of the CFSH was composed of α-helix and connected random coils, as shown by the Alphafold2 results ( Figure 1C).
It was shown by multiple sequence alignments of PtCFSH with other crustacean CFSHs that the predicted PtCFSH protein shares significant identities with other CFSHs in the cleavage site "KR", including "Cysteine" in the interleukin-17 domain (Figure 2A). Furthermore, it was revealed in phylogenetic analysis that the PtCFSH was more closely related to the other CFSH, while it was divided into two branches with subtype CFSH2 ( Figure 2B). The transcript levels of PtCFSH were measured in different tissues obtained from the adult male. It was demonstrated by these results that the PtCFSH transcripts were mainly expressed in the eyestalk, with a slight expression in the brain and testis (Supplementary Figure S1).  It was shown by multiple sequence alignments of PtCFSH with other crustacean CFSHs that the predicted PtCFSH protein shares significant identities with other CFSHs in the cleavage site "KR", including "Cysteine" in the interleukin-17 domain ( Figure 2A). Furthermore, it was revealed in phylogenetic analysis that the PtCFSH was more closely related to the other CFSH, while it was divided into two branches with subtype CFSH2 ( Figure 2B). The transcript levels of PtCFSH were measured in different tissues obtained from the adult male. It was demonstrated by these results that the PtCFSH transcripts were mainly expressed in the eyestalk, with a slight expression in the brain and testis (Supplementary Figure S1).   Most of the CFSH sequences were borrowed from previous works by [21]; other sequences were shown in Table 2.
Cleavage site "KR" is marked with a green box. The eight cysteines are marked in red and the interleukin-17 domain is marked with a blue underline. (B) Phylogenetic tree of CFSHs in crustaceans. Phylogenetic analysis was conducted by the neighbor-joining method in MEGA7. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) was shown next to the branches. PtCFSH was indicated by a red dot.

A B
PtrCFSH Interleukin-17 Domain Most of the CFSH sequences were borrowed from previous works by [21]; other sequences were shown in Table 2.
Cleavage site "KR" is marked with a green box. The eight cysteines are marked in red and the interleukin-17 domain is marked with a blue underline. (B) Phylogenetic tree of CFSHs in crustaceans. Phylogenetic analysis was conducted by the neighbor-joining method in MEGA7. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) was shown next to the branches. PtCFSH was indicated by a red dot.

Effects of PtCFSH on AG
In order to clarify the inhibitory effect of the CFSH on the AG, dsPtCFSH and rPtCFSH were used to conduct the in vivo and in vitro experiments (Figures 3-5). Treatments with dsPtCFSH led to a significant down-regulation of PtCFSH gene expression in the eyestalk, both in vivo and in vitro, showing a high interference efficiency (Figures 3A and  5A). The long-term silencing in PtCFSH in vivo caused an increase in the ratio of type I AG cells and a decrease in type II AG cells, while the injection of rPtCFSH showed an opposite effect ( Figure 3E,F and 4D,E). The types of AG cells were classified according to Sroyraya et al. [28]. Briefly, the type I AG cells have a small globular nucleus containing mainly heterochromatin, while the type II cells are vacuolated with a large cell diameter, containing mostly euchromatin and prominent ribosomes. It was found that the type I cells synthesize IAG in a greater amount than the type II cells [28]. Consistent with the change in the IAG-producing cells, qPCR analysis showed that the PtIAG expression could be reduced by rPtCFSH injection (Figure 4A), while induced by PtCFSH silencing ( Figure 3B). Similar results were also observed in in vitro assays ( Figure 5B,C), further confirming the inhibitory role of the CFSH on IAG expression. ( Figure 3B). Similar results were also observed in in vitro assays ( Figure 5B,C), further confirming the inhibitory role of the CFSH on IAG expression.

Effects of PtCFSH on Testis
To investigate the function of PtCFSH on the testicular development of P. trituberculatus, the histological features of the testis and the expression of spermatogenesis-related genes were evaluated after the in vivo PtCFSH silencing and rPtCFSH injection. It was shown that the treatment of rPtCFSH delayed the further conversion of spermatocytes to spermatids, when compared to the controls ( Figure 4F,G). In contrast, a gradual transformation of spermatocytes to spermatids in the testis was observed in the PtCFSH silencing group ( Figure 3G,H), in parallel with a gradual production of CTFM ( Figure 3H). Meanwhile, the mRNA expression of spermatogenesis-related genes in the testis, such as the kinesinlike protein (Kifc-1), ATP-dependent RNA helicase (Vasa), CyclinB and cyclin-dependent kinase-2 (Cdc-2) [29], was reduced by rPtCFSH injection and induced by PtCFSH silencing (Figures 3C and 4B). The combined results suggest that PtCFSH negatively regulates the spermatogenesis of P. trituberculatus.

Effects of PtCFSH on Testis
To investigate the function of PtCFSH on the testicular development of P. trituberculatus, the histological features of the testis and the expression of spermatogenesis-related genes were evaluated after the in vivo PtCFSH silencing and rPtCFSH injection. It was shown that the treatment of rPtCFSH delayed the further conversion of spermatocytes to spermatids, when compared to the controls ( Figure 4F,G). In contrast, a gradual transformation of spermatocytes to spermatids in the testis was observed in the PtCFSH silencing group ( Figure 3G,H), in parallel with a gradual production of CTFM ( Figure 3H). Meanwhile, the mRNA expression of spermatogenesis-related genes in the testis, such as the kinesin-like protein (Kifc-1), ATP-dependent RNA helicase (Vasa), CyclinB and cyclin-dependent kinase-2 (Cdc-2) [29], was reduced by rPtCFSH injection and induced by PtCFSH silencing (Figures 3C and 4B). The combined results suggest that PtCFSH negatively regulates the spermatogenesis of P. trituberculatus. To find out whether the negative regulatory effect of the CFSH is achieved through IAG, the changes in expression levels of several classical insulin pathway genes, including IAG itself, insulin-like growth factor banding protein-related peptide (IGFBP-rp), insulinlike hormone receptor 1 (IR1), IR2, protein kinase B (Akt), mammalian target of rapamycin (mTOR), and forkhead transcription factors (Foxo) [30,31], were further determined in the testis in the two treatments. In addition, since the insulin pathway activates the phosphorylation of MAPK [32], the change in the phosphorylated MAPK levels were also examined using Western blotting. As expected, the expression of insulin pathway genes and phosphorylated MAPK levels were reduced by rPtCFSH injection and induced by PtCFSH silencing (Figure 3C,D and Figure 4B,C). Therefore, it was speculated that the CFSH can inhibit the production of IAG, which, in turn, affects the testicular development.
Furthermore, in vitro experiments were conducted to explore whether the CFSH had a direct regulatory effect on the testis. It was found that the addition of rPtCFSH into the testis explants also led to a decrease in spermatogenesis-related gene expression and the MAPK phosphorylation level, which means that the CFSH could act directly on the testis ( Figure 5D,E). Meanwhile, it was also observed that the expression levels of IAG and insulin pathway genes in the testis were down-regulated ( Figure 5D).

Validation of the CFSH-IAG-Testis Endocrine Axis
A short-term RNAi experiment was performed to verify the existence of the CFSH-IAG-testis endocrine axis. Similar changes in the levels of gene expression and MAPK phosphorylation were observed in short-term injection with dsPtCFSH, when compared to those in long-term treatment ( Figure 6A,D). Opposite effects were found in the dsPtIAG injection group (Figure 6B,D), indicating the stimulatory role of IAG in testicular development. To confirm the central role of IAG in this endocrine axis, a mixed injection of dsPtCFSH and dsPtIAG was further performed, and the results were similar to those of the dsPtIAG injection alone ( Figure 6C,D). It can be understood that the regulatory effect of dsPtCFSH on the testicular development was blocked after the injection of dsPtIAG, which indicated the existence of the CFSH-IAG-testis endocrine axis. Different colors of the same tested gene between the experimental and control groups indicated significant differences. The darker the color in the heat map, the higher the relative expression of the tested genes and vice versa. (B) Heat map expression levels of related genes after injection of dsPtIAG, dsGFP. Different colors of the same tested gene between the experimental and control groups indicated significant differences. The darker the color in the heat map, the higher the relative expression of the tested genes and vice versa. (C) Heat map expression levels of related genes after injection of dsPtIAG followed by dsPtCFSH, dsGFP. Different colors of the same tested gene between the experimental and control groups indicated significant differences. The darker the color in the heat map, the higher the relative expression of the tested genes and vice versa. (D) Changes in phosphorylated-MAPK expression levels after injection of dsPtIAG followed by dsPtCFSH, dsPtCFSH, and dsGFP. Total-MAPK, β-tubulin as internal reference proteins. The scale bar is shown in the lower right corner. Vertical bars represent means ± SEM (n = 5). Significant differences among stages are indicated by different letter labels (one-way ANOVA followed by post hoc Tukey's multiple group comparison p < 0.05).

Possible Signaling Mechanisms of PtCFSH
Most neuropeptides use the G protein-coupled receptor (GPCR) as their membrane receptor, and the signaling system needs the participation of second messengers [27]. To explore which second messengers are involved in the signal transduction of PtCFSH, the levels of cAMP and cGMP, as well as the activity of nitric oxide synthetase (NOS), were determined in in vitro testis experiments. Remarkable down-regulation at 5 min and 15 min after adding rPtCFSH was shown by both cAMP and cGMP levels compared to the control group ( Figure 7A,B). In addition, the NOS enzymatic activity was significantly diminished after adding rPtCFSH, suggesting a decrease in the NO level ( Figure 7C). Different colors of the same tested gene between the experimental and control groups indicated significant differences. The darker the color in the heat map, the higher the relative expression of the tested genes and vice versa. (B) Heat map expression levels of related genes after injection of dsPtIAG, dsGFP. Different colors of the same tested gene between the experimental and control groups indicated significant differences. The darker the color in the heat map, the higher the relative expression of the tested genes and vice versa. (C) Heat map expression levels of related genes after injection of dsPtIAG followed by dsPtCFSH, dsGFP. Different colors of the same tested gene between the experimental and control groups indicated significant differences. The darker the color in the heat map, the higher the relative expression of the tested genes and vice versa. (D) Changes in phosphorylated-MAPK expression levels after injection of dsPtIAG followed by dsPtCFSH, dsPtCFSH, and dsGFP. Total-MAPK, β-tubulin as internal reference proteins. The scale bar is shown in the lower right corner. Vertical bars represent means ± SEM (n = 5). Significant differences among stages are indicated by different letter labels (one-way ANOVA followed by post hoc Tukey's multiple group comparison p < 0.05).

Possible Signaling Mechanisms of PtCFSH
Most neuropeptides use the G protein-coupled receptor (GPCR) as their membrane receptor, and the signaling system needs the participation of second messengers [27]. To explore which second messengers are involved in the signal transduction of PtCFSH, the levels of cAMP and cGMP, as well as the activity of nitric oxide synthetase (NOS), were determined in in vitro testis experiments. Remarkable down-regulation at 5 min and 15 min after adding rPtCFSH was shown by both cAMP and cGMP levels compared to the control group ( Figure 7A,B). In addition, the NOS enzymatic activity was significantly diminished after adding rPtCFSH, suggesting a decrease in the NO level ( Figure 7C).

Discussion
The CFSH is a primary hormone involved in developing relevant phenotypes in female crustaceans [16,22,23,33]. However, the regulatory mechanisms of CFSH in male crustaceans have been under investigated. The full-length CFSH gene sequence of P. trituberculatus, encoding 225 amino acids, including the signal peptide, a cleavage KR site, and an IL-7 structural domain containing eight cysteines, was investigated in this study. It was shown that it has the same structural organization as type I CFSHs. Multiple sequence comparisons and a phylogenetic tree analysis supported that PtCFSH belongs to type I CFSHs (Figures 1 and 2). Most type I CFSHs contain eight cysteines [17][18][19][20][21][22]34,35], which play a role in forming the disulfide bonds necessary for their normal biological function [21,36,37]. In crustaceans, it has been reported in various species that it is exclusively expressed in the eyestalk, such as CsaCFSH, SpCFSH, and LvCFSH1a, which have been shown to stimulate female sexual differentiation [16,22,33]. In contrast, MrCFSH1a/b and MajCFSH were detected in several distinct tissues. Similar results were also observed in the present study, showing that, in parallel to the strong signal detected in the eyestalk, PtCFSH also has a slight signal within the testicular tissue (Supplementary Material). Thus, the CFSH may play a more extensive role in decapod crustaceans [19,21].
In the present study, the physiological role of PtCFSH in male P. trituberculatus was investigated in a series of in vivo and in vitro experiments, based on the dsPtCFSH RNA silencing and rPtCFSH treatment. Consistent with the reported negative regulation of IAG by CFSH [22,23], PtCFSH silencing and rPtCFSH treatment both suggested that PtCFSH can significantly inhibit the expression level of PtIAG in vitro and in vitro. Furthermore, the histological study on the AG showed that PtCFSH had a negative effect on the number of IAG-producing cells. This would be reminiscent of the bilateral eyestalk ablation of the P. pelagicus that caused an increase in the cells producing IAG [28]. In Macrobrachium nip-

Discussion
The CFSH is a primary hormone involved in developing relevant phenotypes in female crustaceans [16,22,23,33]. However, the regulatory mechanisms of CFSH in male crustaceans have been under investigated. The full-length CFSH gene sequence of P. trituberculatus, encoding 225 amino acids, including the signal peptide, a cleavage KR site, and an IL-7 structural domain containing eight cysteines, was investigated in this study. It was shown that it has the same structural organization as type I CFSHs. Multiple sequence comparisons and a phylogenetic tree analysis supported that PtCFSH belongs to type I CFSHs (Figures 1 and 2). Most type I CFSHs contain eight cysteines [17][18][19][20][21][22]34,35], which play a role in forming the disulfide bonds necessary for their normal biological function [21,36,37]. In crustaceans, it has been reported in various species that it is exclusively expressed in the eyestalk, such as CsaCFSH, SpCFSH, and LvCFSH1a, which have been shown to stimulate female sexual differentiation [16,22,33]. In contrast, MrCFSH1a/b and MajCFSH were detected in several distinct tissues. Similar results were also observed in the present study, showing that, in parallel to the strong signal detected in the eyestalk, PtCFSH also has a slight signal within the testicular tissue (Supplementary Material). Thus, the CFSH may play a more extensive role in decapod crustaceans [19,21].
In the present study, the physiological role of PtCFSH in male P. trituberculatus was investigated in a series of in vivo and in vitro experiments, based on the dsPtCFSH RNA silencing and rPtCFSH treatment. Consistent with the reported negative regulation of IAG by CFSH [22,23], PtCFSH silencing and rPtCFSH treatment both suggested that PtCFSH can significantly inhibit the expression level of PtIAG in vitro and in vitro. Furthermore, the histological study on the AG showed that PtCFSH had a negative effect on the number of IAG-producing cells. This would be reminiscent of the bilateral eyestalk ablation of the P. pelagicus that caused an increase in the cells producing IAG [28]. In Macrobrachium nipponense, RNAi in several eyestalk neuropeptides revealed that the gonad-inhibiting hormone (GIH) and molt-inhibiting hormone (MIH) had an inhibitory effect on IAG expression [15]. Our results confirmed that the CFSH is also a neuropeptide with IAG inhibition.
It was revealed in our in vivo studies that rPtCFSH injection could reduce the conversion of spermatocytes to spermatids ( Figure 4F,G), while this process was stimulated when dsCFSH was silenced ( Figure 3G,H). In addition, the expression levels of spermatogenesisrelated genes, such as kifc-1, vasa, cyclinB, and cdc-2 [38,39], changed accordingly in two treatments, suggesting that PtCFSH may be involved in testis development and spermatogenesis in P. trituberculatus. The effects of PtCFSH on testis might be achieved through IAG, since several classical insulin pathway genes, including IAG itself, insulin-like growth factor binding protein-related peptide (IGFBP-rp), insulin-like hormone receptor 1 (IR1), IR2, protein kinase B (Akt), mammalian target of rapamycin (mTOR), and forkhead transcription factors (Foxo) [30,31,40,41], were also induced by PtCFSH silencing and simultaneously reduced by rPtCFSH. As one of the two major intracellular pathways in the conserved insulin signaling system, the MAPK pathway mainly controls development and reproduction [42]. Therefore, the change in testicular MAPK phosphorylation levels in PtCFSH-based manipulations ( Figures 3D and 4C) might give further evidence that IAG is involved in the CFSH-mediated testicular development.
To confirm the existence of the CFSH-IAG-testis endocrine axis, a short-term injection of dsPtIAG was further performed. It was found that this treatment reduced the levels of gene expression and MAPK phosphorylation. Given the inhibitory effect of PtCFSH on IAG expression, dsPtCFSH was also injected in the short-term experiments, with the purpose of causing a transient increase in PtIAG expression; this obtained opposite effects to the PtIAG silencing. In fact, the short-term silencing in PtCFSH confirms the regulatory relationship between PtCFSH with PtIAG and related genes, which had been revealed in the long-term experiments. The co-injection of both dsPtCFSH and dsPtIAG showed similar effects on the testis to the single injection of dsPtIAG; it could be understood that the regulatory effect of dsPtCFSH on the testis was blocked after the injection of dsPtIAG, demonstrating the central role of IAG in connecting the CFSH and testis.
In addition to the indirect effects of PtCFSH on the testis, the in vitro treatment of rPtCFSH on the testis explants suggested that the PtCFSH was able to act directly on the testis. Interestingly, however, this direct effect might also be related to the IAG. Our results showed that rPtCFSH can also inhibit the levels of PtIAG and insulin pathway gene expression in the testis, as well as the MAPK phosphorylation. Although the presence of IAG in testis has been less reported, it was revealed by our previous study that PtIAG is expressed in the testis of P. trituberculatus [43]. Since the paracrine/autocrine is considered to be an important mechanism of crustacean hormonal regulation [44][45][46], the testis-derived IAG might also be involved in the regulation of testicular development. Therefore, the inhibitory effect of the CFSH on IAG expression might not be restricted to the AG, but might also occur in other tissues. One circumstantial piece of evidence may come from studies of the CFSH in females, finding that the CFSH can inhibit the expression of IAG in the ovaries [22].
To exert its physiological functions, whether on the AG or the testis, the CFSH must have its own set of signaling mechanisms. As a neuropeptide, GPCR is a primary candidate for the CFSH receptor [27]. However, since numerous types of GPCRs exist [33], the receptors for the CFSH has not been firmly validated. Among the signaling pathways regulated by GPCRs, the second messenger is particularly important, and cAMP, cGMP, and NO are the most common ones [47]. Therefore, we examined the level changes in these second messengers in testis explants after rPtCFSH treatment. It was found that the level of cAMP and cGMP, as well as the NOS activity, were all down-regulated by rPtCFSH. This would be reminiscent of the signaling system deduced for MIH, which is constituted of a "triggering phase" involving cAMP/Ca 2+ pathway, a "summation phase" involving the NO/cGMP pathway, and the calmodulin (CaM), which links the two phases by activating NOS [27]. It is likely that the CFSH uses a similar system for its signal transduction. Furthermore, these results also imply that the PtCFSH might act on the testis, independent of IAG.

Conclusions
In conclusion, the P. trituberculatus CFSH was identified, cloned and expressed in the recombinant form. The physiological functions of PtCFSH on the AG and testis were investigated using the RNAi and recombinant protein injection in a series of in vivo and in vitro experiments. It was confirmed that PtCFSH can negatively regulate the spermatogenesis and testicular development via a CFSH-IAG-testis endocrine axis. PtCFSH may also act directly on the testis, dependent or independent of IAG. The signaling system of PtCFSH involved the participation of cAMP, cGMP, and NO, which resembles the MIH signaling pathway. In order to summarize, a hypothetical pathway for the mode of the CFSH action in P. trituberculatus was constructed by figdraw ( Figure 8).

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In conclusion, the P. trituberculatus CFSH was identified, cloned and expressed in the recombinant form. The physiological functions of PtCFSH on the AG and testis were investigated using the RNAi and recombinant protein injection in a series of in vivo and in vitro experiments. It was confirmed that PtCFSH can negatively regulate the spermatogenesis and testicular development via a CFSH-IAG-testis endocrine axis. PtCFSH may also act directly on the testis, dependent or independent of IAG. The signaling system of PtCFSH involved the participation of cAMP, cGMP, and NO, which resembles the MIH signaling pathway. In order to summarize, a hypothetical pathway for the mode of the CFSH action in P. trituberculatus was constructed by figdraw ( Figure 8). Hypothetical pathway for the mode of the CFSH action in P. trituberculatus. There may be different pathways for CFSH. One is acting on the testis through the classical insulin pathway and the other is acting directly as a neuropeptide to complete the signal transduction. IAG: insulin-like androgenic gland hormone; IGFBP-rp: insulin growth factor binding protein-related protein; IR: insulin-like hormone receptor; IRS: Insulin receptor substrate; G: G protein; AC: activation of adenylyl cyclase; GC-I: NO-sensitive guanylyl cyclase.

Supplementary Materials:
The following supporting information can be downloaded at: www.mdpi.com/xxx/s1, Figure S1: Detection of PtCFSH expression in tissues by RT-PCR; Figure S2: Prokaryotic expression and purification of PtCFSH and empty vector; Figure S3: Phosphorylated-MAPK was detected in the testis after long-term in vivo injection of rPtCFSH; Figure S4: Phosphorylated-MAPK was detected in testis after long-term in vivo injection of dsPtCFSH, dsGFP; Figure  S5: Phosphorylated-MAPK was detected in the testis after incubation of testis in vitro and addition of rPtCFSH and rGST, respectively; Figure S6: Phosphorylated-MAPK was detected in testis after short-term in vivo injection of dsCFSH, dsIAG, dsIAG followed by dsCFSH, and dsGFP, respectively; Table S1: Primers used in this study; Table S2: List of species used in phylogenetic analyses and multiple sequence comparisons.
Author Contributions: M.W. X.X. and D.Z. designed the study; M.W and R.X. performed the experiments; S.T. and Q.Y. analyzed the data; M.W wrote the manuscript; X.X. and D.Z. revised the manuscript. All authors have read and agreed to the published version of the manuscript.
Funding: This study was supported by the National natural Science Foundation of China (Nos. Hypothetical pathway for the mode of the CFSH action in P. trituberculatus. There may be different pathways for CFSH. One is acting on the testis through the classical insulin pathway and the other is acting directly as a neuropeptide to complete the signal transduction. IAG: insulin-like androgenic gland hormone; IGFBP-rp: insulin growth factor binding protein-related protein; IR: insulin-like hormone receptor; IRS: Insulin receptor substrate; G: G protein; AC: activation of adenylyl cyclase; GC-I: NO-sensitive guanylyl cyclase.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ani13040690/s1, Figure S1: Detection of PtCFSH expression in tissues by RT-PCR; Figure S2: Prokaryotic expression and purification of PtCFSH and empty vector; Figure S3: Phosphorylated-MAPK was detected in the testis after long-term in vivo injection of rPtCFSH; Figure S4: Phosphorylated-MAPK was detected in testis after long-term in vivo injection of dsPtCFSH, dsGFP; Figure S5: Phosphorylated-MAPK was detected in the testis after incubation of testis in vitro and addition of rPtCFSH and rGST, respectively; Figure S6: Phosphorylated-MAPK was detected in testis after short-term in vivo injection of dsCFSH, dsIAG, dsIAG followed by dsCFSH, and dsGFP, respectively; Table S1: Primers used in this study; Table S2: List of species used in phylogenetic analyses and multiple sequence comparisons.
Author Contributions: M.W., X.X. and D.Z. designed the study; M.W. and R.X. performed the experiments; S.T. and Q.Y. analyzed the data; M.W. wrote the manuscript; X.X. and D.Z. revised the manuscript. All authors have read and agreed to the published version of the manuscript.